Journal of Experimental Marine Biology and Ecology 332(1)
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Journal of Experimental Marine Biology and Ecology 332 (2006) 27–36 www.elsevier.com/locate/jembe Chemical and physical defenses against predators in Cystodytes (Ascidiacea) Susanna Lo´pez-Legentil a,b,*, Xavier Turon a, Peter Schupp c a Department of Animal Biology (Invertebrates), Faculty of Biology, University of Barcelona, 645 Diagonal Ave., 08028 Barcelona, Spain b Laboratoire de Chimie des Biomole´cules et de l’Environnement (Centre de Phytopharmacie), University of Perpignan, 52 Paul Alduy Ave., 66860 Perpignan, France c Marine Laboratory, University of Guam, UOG Station, Mangilao, GU 96923, Guam, USA Received 4 July 2005; received in revised form 1 November 2005; accepted 1 November 2005 Abstract Ascidians utilize both physical (spicules, tunic toughness) and chemical defenses (secondary metabolites, acidity) and suffer relatively little predation by generalist predators. The genus Cystodytes (Polycitoridae) is distributed widely in both tropical and temperate waters. Secondary metabolite composition, calcareous spicules and tunic acidity (pHb1) may act as redundant defense mechanisms against predation in this genus. To assess the relative importance of chemical and physical defenses against predation in ascidians, we studied purple and blue morphs of Cystodytes from the western Mediterranean (formerly assigned to Cystodytes dellechiajei, but recently shown to belong to two different species), and a purple morph from Guam (USA), identified as Cystodytes violatinctus. Crude extracts, spicules, ascididemin (the major alkaloid of the blue morph) and acidity were used in feeding trials to evaluate chemical and physical defense mechanisms in Cystodytes spp. We performed feeding experiments in the field with a guild of generalist fish (mostly damselfish), and in the laboratory with a sea urchin and a puffer fish. Our results showed that all crude extracts and ascididemin significantly deterred fish predation, but not sea urchin predation. However, neither acidity alone nor spicules at natural concentrations deterred feeding. These results and other studies on sponges and gorgonians suggest that secondary metabolites are the primary means of defense against fish predators. Spicules and tunic acidity may perform other ecological roles and/or target certain specialist predators. D 2005 Elsevier B.V. All rights reserved. Keywords: Acidity; Ascidians; Ascididemin; Cystodytes; Defense mechanisms; Deterrence; Predation; Toxicity 1. Introduction range of defense mechanisms, including behavioral (e.g. mimicry), physical (e.g. spicules, tissue toughness) Benthic marine invertebrates are under intense com- and chemical (e.g. bioactive secondary metabolites, petitive pressure for space, light and nutrients. In addi- acid pH) strategies, to ensure survival. The relative tion, sessile organisms are easy prey for predators. importance and potential interactions of different de- Many of these organisms, therefore, have developed a fense mechanisms may depend on: the organism exam- ined (Pennings and Paul, 1992; Hay et al., 1994), the inability of a single defense to deter all type of pre- * Corresponding author. Present address: Biological Sciences and Center for Marine Science, University of North Carolina at Wilming- dators and/or competitors (Paul and Hay, 1986; Hay et ton, 5600 Marvin K. Moss Lane, Wilmington, NC 28409, USA. al., 1987; Schupp and Paul, 1994; Pisut and Pawlik, E-mail address: [email protected] (S. Lo´pez-Legentil). 2002; Tarjuelo et al., 2002; Burns et al., 2003), the 0022-0981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2005.11.002 28 S. Lo´pez-Legentil et al. / Journal of Experimental Marine Biology and Ecology 332 (2006) 27–36 organism’s life history stage (Uriz et al., 1996; Pisut predation (Parry, 1984; Tarjuelo et al., 2002). All in all, and Pawlik, 2002), the developmental or physiological little experimental evidence exists regarding the inter- constraints that might prevent a particular defense from action of chemical and physical defense mechanisms in being used in all parts of the organism, necessitating ascidians. additional defenses to ensure protection of the whole We chose the widely distributed ascidian genus organism (Harvell and Fenical, 1989), evolutionarily Cystodytes (Aplousobranchiata: Polycitoridae) to assess established constraints (Schmitt et al., 1995; Kubanek the relative importance of physical and chemical et al., 2002) and whether structures or compounds are defenses against predation in ascidians. Lo´pez-Legentil energetically expensive, among other factors. For in- et al. (2005a) identified two chemotypes within Medi- stance, in some sponges, both large spicules and chem- terranean specimens of this genus, previously attributed ical defenses deter fish feeding (Pawlik et al., 1995; to Cystodytes dellechiajei (Della Valle, 1877). Genetic O’Neal and Pawlik, 2002; Burns et al., 2003; Burns and and biological evidence indicates that these two che- Ilan, 2003). Soft corals and gorgonians produce both motypes correspond to sibling species (Lo´pez-Legentil chemical defenses and sclerites, which, in some cases, and Turon, 2005; Lo´pez-Legentil et al., 2005b). The protect them against predators (Pawlik et al., 1987; first chemotype was characterized by the presence of Harvell et al., 1988; Wylie and Paul, 1989; Pawlik C9-unsubstituted pyridoacridines such as ascididemin and Fenical, 1992; Puglisi et al., 2000, 2002; O’Neal (Fig. 1; Kobayashi et al., 1988) and 11-hydroxyascidi- and Pawlik, 2002). However, the primary function of demin (Schmitz et al., 1991), which were present in sclerites may be structural rather than defensive (Koehl, blue and green morphs from the western Mediterra- 1982; Lewis and Von Wallis, 1991). In view of these nean. The second chemotype contained the sulfur-con- and similar results, Van Alstyne et al. (1994) suggested taining pyridoacridines kuanoniamine D (Caroll and that, when feeding experiments are designed, all de- Scheuer, 1990), shermilamine B (Carroll et al., 1989) fense mechanisms against predation that an organism and their deacetylated forms (Eder et al., 1998; and might use should be tested in combination, rather than Lo´pez-Legentil et al., 2005a). These pyridoacridines each single defense separately. However, mainly due to were present in a purple morph from the western Med- experimental limitations, few studies have attempted iterranean, and were also found in a purple morph from this (Paul and Van Alstyne, 1992; Schupp and Paul, Guam, identified as Cystodytes violatinctus Monniot, 1994; O’Neal and Pawlik, 2002; Hill et al., 2005). 1988 (Lo´pez-Legentil, 2005). Some of these substances Among benthic invertebrates, ascidians suffer rela- are highly cytotoxic (Eder et al., 1998; Dassonneville et tively little predation by generalist predators (Millar, al., 2000; Bowden, 2000). Ascididemin also has some 1971; Goodbody and Gibson, 1974; Stoecker, 1980a). antibacterial and antifungal action (Lindsay et al., These generalist predators are mainly fish (Randall and 1995). To our knowledge, apart from a possible anti- Hartman, 1968; Myers, 1983), and occasionally urchins fouling function (Debard et al., 1998), no ecological (Briscoe and Sebens, 1988). Specialist predators on role has been described for ascididemin. In addition to ascidians include mollusks, such as the lamellarians, the bioactive compounds cited above, Cystodytes spp. cypraeids (Fretter and Graham, 1962; Millar, 1971; colonies are highly acidic (pHb2; Parry, 1984; Tarjuelo Lambert, 1980) and nudibranchs (Millar, 1971; Paul et al., 2002) and contain calcareous spicules encasing et al., 1990), and polyclad flatworms (Millar, 1971; the zooids (Turon, 1987; Kott, 1990). These potential Morris et al., 1980; Schupp et al., 1999; Caralt et al., defenses seem to result in an effective defense mecha- 2002). In addition, ascidians have both physical (spicules, 2 1 tunic toughness) and chemical defenses (Swinehart et 3 al., 1974; Stoecker, 1978, 1980b; Pisut and Pawlik, N 2002; Tarjuelo et al., 2002). Numerous studies support 4 N the deterrent role of secondary metabolites (Paul et al., 5 1990; Davis, 1991; McClintock et al., 1991; Lindquist 6 et al., 1992; Vervoort et al., 1998). High vanadium 9 8 concentrations (Stoecker, 1980b) and low pH (Webb, N 7 1939; Stoecker, 1978, 1980a,b,c; Pisut and Pawlik, 2002) have also been suggested as anti-predatory O defenses. However, other studies indicated that neither Fig. 1. Chemical structure of ascididemin, the major alkaloid of the the presence of vanadium nor an acidic pH prevented Mediterranean blue morph. S. Lo´pez-Legentil et al. / Journal of Experimental Marine Biology and Ecology 332 (2006) 27–36 29 nism (although their relative importance and interac- were weighed and freeze-dried. They were then tions are not known), because Cystodytes spp. colonies extracted three times in a 1:1 (v/v) mixture of dichlor- generally lack epibionts and show scarce signs of pre- omethane and methanol and the combined extracts were dation. As the vanadium concentration in Cystodytes concentrated under vacuum to leave a powdery organic (V10 ppm dry weight; Stoecker, 1980b) is well below residue. The mean natural concentration of crude ex- the concentration suggested to deter predation in other tract found was 2.01% (F0.21 S.E.M., N =5 for each ascidians (N1000 ppm dry weight; Stoecker, 1980b), it color morph) per wet mass of the ascidian. We used this will be ignored in the present study. Tarjuelo et al. concentration, relative to